Identifying substances using cells that express p75NTR

ABSTRACT

Methods for evaluating the risk of an individual to develop Alzheimer&#39;s disease using cultured neural crest-derived melanocytes are described. Also described are methods of therapy for Alzheimer&#39;s disease using peptides that bind to the neurotrophin receptor (p75 NTR ) and competitively inhibit the binding of β-amyloid to the p75 NTR .

RELATED APPLICATIONS

This application is a continuation of Ser. No. 09/163,095, filed Sep.29, 1998, now U.S. Pat. No. 6,242,416 which is a continuation-in-part ofPCT/US97/04966, filed Mar. 28, 1997, which is a continuation-in-part ofprior Ser. No. 08/625,765 filed Mar. 29, 1996, now abandoned, the entireteachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Dementia is a condition of deteriorating mentality that is characterizedby marked decline in the individual's former intellectual level,including memory loss, impaired judgment, speech and orientation, and isoften accompanied by emotional apathy. (WEBSTER'S MEDICAL DESKDICTIONARY, Merriam-Webster, Inc., Springfield, Mass., p.169 (1986)).

A leading cause of dementia is Alzheimer's disease,(AD), aneurodegenerative disorder affecting 17 to 20 million people worldwide(Yamazaki, T., et al., J. Cell. Biol., 129;431-442 (1995); Brinaga, M.,Science, 269:917-918 (1995); Lavy-Lahad, E., et al., Science,269:970-972 (1995); Lavy-Lahad, E., et al, Science, 269:973-977 (1995)).AD is characterized by progressive dementia together withneuropathological findings of “senile plaques” in the brain formed bydeposits of β-amyloid protein, surrounded by clusters of degeneratingneurons. β-amyloid protein itself is a fragment of the 770 amino acidmembrane bound β-amyloid precursor protein (βAPP) that is expressed inboth neuronal and non-neuronal tissues.

Muteins of βAPP have been produced for the purpose of developing a βAPPsubstrate system wherein βAPP is cleavable or not cleavable such thatβAP producing enzymes and inhibitors thereof may be isolated.

The specific cause of Alzheimer's disease has not yet been determined. Amutation in the βAPP gene in families with one form of autosomaldominant AD was found to be associated with increased β-amyloidsynthesis and aggregation in the brain. A receptor for βAPP has beenidentified as the low density lipoprotein receptor-related protein,ApoE, and it has been postulated that this receptor protein, the enzymeresponsible for βAPP cleavage in the cell membrane, production of βAPPand/or production of extracellular matrix molecules may be abnormalindividually or in combination in AD patients, resulting in excessβ-amyloid deposition and the observed neurotoxicity. However, themechanism by which other known βAPP gene mutations cause AD, as well asthe pathophysiology of non-familial AD in which βAPP gene mutations havenot been recognized, is not understood.

Therefore, diagnosing Alzheimer's disease as the cause of anindividual's dementia, as well as treating AD and developing drugtherapies is very difficult. Although recent reports of usingPositron-emission tomography (PET) (Reiman, E. M., et al., New Eng. JMed., 334:752-758 (1996), determining the genotype of an individual'sApoE, or measuring the levels of β-amyloid protein in cerebral spinalfluid may be promising, diagnosis of Alzheimer's is currently confirmedonly upon autopsy to determine the presence of β-amyloid senile plaques.

In vitro systems employed to study Alzheimer's disease to date consistof malignant, or transformed cells that are not of neural crest origin,or lower vertebrate neuronal cultures. It would be of great advantage tohave an Alzheimer's disease model system using normal human neuralcrest-derived cells. However, to date, no such model system has beendeveloped.

Moreover, recent studies have shown that damage to CNS neurons due toAlzheimer's disease begins years before clinical symptoms are evident.(Reiman, E. M., et al., New Eng. J Med., 334:752-758 (1996)), suggestingthat therapy could begin in the pre-symptomatic phase of the disease ifa sensitive diagnostic test and targeted therapies were available. Thereexists a great need to determine the physiological mechanisms involvedwith the disease and for an accurate and easy to perform assay toevaluate the risk of developing Alzheimer's disease.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that neurons andepidermal melanocytes are neural crest-derived cells that undergoβ-amyloid mediated apoptosis mediated by β-amyloid binding to the samereceptor, the 75 kD neurotrophin receptor (p75^(NTR)). As used herein,the term β-amyloid protein is intended to encompass β-amyloid protein (a4.2 kD polypeptide (Selkoe, D. J., Neuron, 6:487-498 (1991); Glenner G.G. and Wong, C. W., Biochem. Biophys. Res. Commun., 120:885-890 (1993),the teachings of which are herein incorporated by reference), β-amyloidprecursor protein (βAPP), and fragments of β-amyloid and β-amyloidprecursor protein referred to herein as β-amyloid peptides, includingβ-amyloid 1-40 peptide, β-amyloid 1-42 peptide, β-amyloid 25-36 peptideor β-amyloid 28-30 peptide. (β-amyloid protein is also referred toherein as β-amyloid).

More specifically, it is demonstrated herein that β-amyloid protein orpeptide binds to the p75 nerve growth factor receptor (p75^(NTR)) ofneural crest-derived cells, e.g. melanocytes, resulting in apoptosis ofthe cell. It is further demonstrated herein that inhibiting the bindingof β-amyloid protein or peptide to the p75 nerve growth factor receptor,results in inhibiting the activation of the p75 nerve growth factorreceptor, which in turn inhibits apoptosis.

The present invention relates to methods of inhibitingβ-amyloid-mediated activation of the p75 nerve growth factor receptor ofa cell that expresses the p75 nerve growth factor receptor, methods ofinhibiting the binding of β-amyloid protein and β-amyloid peptides tothe p75 nerve growth factor receptor, and methods of inhibitingβ-amyloid-mediated apoptosis of neural crest-derived cells. The methodscomprise contacting the cell with a substance, comprising, for example,the amino acid sequence lysine-glycine-lysine (KGK) orlysine-glycine-alanine (KGA), wherein the substance binds to the p75nerve growth factor receptor, resulting in the inhibition of β-amyloidprotein or β-amyloid peptide binding to and/or activation of the p75nerve growth factor receptor, or wherein the substance inhibitsβ-amyloid protein or β-amyloid peptide mediated apoptosis of the cellwhich expresses the p75 nerve growth factor receptor.

It is has been reported that cell death receptors mediate apoptosis byaggregation resulting from ligand binding or membrane perturbation.Applicants have demonstrated that β-amyloid aggregates the p75^(NTR)thereby inducing cell death. Peptides, specifically cyclic peptides,bind the receptors individually and block aggregation of the p75^(NTR),thereby inhibiting apoptosis. Specifically encompassed by the presentinvention are cyclic peptides (e.g., peptides in a β-loop conformation)which comprise lysine-glycine-lysine or lysine-glycine-alanine, or othersequences capable of binding to the p75^(NTR.)

Activation of the p75 nerve growth factor receptor can be determined bymeasuring the β-amyloid activation of the p75 nerve growth factorreceptor of neural crest-derived cells, in culture or in a tissuesample, in the presence of the test substance and comparing the resultswith the β-amyloid activation of the p75 nerve growth factor receptor ofneural crest-derived cells in a control culture or sample without thetest-substance. A decrease of β-amyloid activation of the p75 nervegrowth factor receptor of neural crest-derived cells in the test samplecompared to β-amyloid activation of the p75 nerve growth factor receptorof neural crest-derived cells in the control sample is indicative of asubstance that inhibits β-amyloid-mediated apoptosis in neuralcrest-derived cells.

The present invention further relates to in vitro methods of screeningsubstances and identifying those substances capable of inhibiting, ordecreasing cell apoptosis mediated by β-amyloid, or activation of thep75^(NTR), and to substances identified by these methods.

The method of identifying substances that inhibit β-amyloid-mediatedapoptosis of cells that express the p75 nerve growth factor receptorcomprises contacting the cells, in culture or in a tissue sample, withβ-amyloid protein, or peptide and with the substance to be tested,wherein the β-amyloid protein/peptide is at a concentration sufficientto bind to and activate the p75 nerve growth factor receptor, therebyproducing a test sample. The test sample is maintained under conditionssufficient for the β-amyloid protein or peptide or test-substance tobind to the p75 nerve growth factor receptor expressed on the cells andactivation of the p75 nerve growth factor receptor is determined,thereby identifying substances that inhibit apoptosis.

The present invention further relates to methods of diagnosing andtreating Alzheimer's disease, and other neurodegenerative diseasesmediated by β-amyloid protein, or by aberrant activation of the lowaffinity nerve growth factor receptor localized on neural cell surfaces.For example, autoimmune encephalomyelitis, Huntington's disease, Pick'sdisease, corticobasal degeneration, progressive supra-nuclear palsy,Gerotman-Shausslesr Scheinker syndrome, Niemann-Pick disease, Down'sSyndrome and progressive supranuclear palsy are encompassed by thisinvention.

Neurodegenerative diseases mediated by β-amyloid protein encompassdiseases affecting neural crest-derived cells, such as central nervoussystem (CNS) neurons and in which β-amyloid protein, PAPP, or β-amyloidpeptides initiate, or exacerbate, a process leading to neurondegeneration, or neuronal cell death. Neurodegenerative diseasesmediated by aberrant activation of the low affinity nerve growth factorencompasses diseases in which the low affinity nerve growth factor isactivated by a substance resulting in apoptotic cell death. Theneurodegenerative diseases encompassed by the present invention arecharacterized by progressive dementia in the affected individual.Specifically encompassed by the present invention is theneurodegenerative disease, Alzheimer's disease (AD), which ischaracterized by the deposition of -amyloid peptides in neural tissue,leading to neuronal cell degeneration, cell death and progressivedementia.

The present invention also relates to methods of evaluating the risk ofan individual to develop Alzheimer's disease using an in vitro assaysystem comprising epidermal melanocytes obtained from the individual.Epidermal melanocytes are melanocytes found in the epidermis (skin) andhair bulbs of vertebrates. The present invention is based on Applicants'findings that human melanocytes exhibit significant similarities withcentral nervous system neurons (the cells predominantly affected byAlzheimer's disease) and that melanocytes utilize the same signalingmolecules as neurons to determine their survival versus programmed celldeath (apoptosis).

For example, neuronal cells express a high affinity (p140^(trkA)) and alow affinity receptor (p75^(NTR)) for nerve growth factor (NGF). Asdescribed herein, Applicants have demonstrated that these nerve growthfactor receptors are also expressed on melanocytes and that β-amyloidbinds to the low affinity nerve growth factor receptor, p75^(NTR),expressed on the melanocyte surface. Applicants also demonstrate hereinthat binding of β-amyloid to the p75^(NTR) activates the receptor,resulting in apoptotic cell death of the melanocytes. Applicants furtherdemonstrate that the β-amyloid mediated-apoptosis can be competitivelyblocked by providing nerve growth factor or a biologically activefragment, analog or derivative thereof. Nerve growth factor is aphysiologic ligand for p75^(NTR) that has a 2-3-fold higher receptoraffinity than β-amyloid and when binding coordinately to p75^(NTR) andp140^(trkA), exhibits a 200-fold higher affinity

Additionally, Applicants have demonstrated that melanocytes secreteβ-amyloid (in the form of its precursor protein) constitutively, and inincreased amounts in response to trauma, e.g., UV irradiation. Thus,based on results described herein,

Applicants have established that melanocytes, easily obtainable fromskin biopsies, are reasonable model cells for the study and diagnosis ofAlzheimer's disease.

Based on the results described herein, neuronal cell death resulting inAlzheimer's Disease can arise in several different settings. Forexample, if an excess of β-amyloid protein is present in the centralnervous system (CNS), more β-amyloid protein will bind to the p75^(NTR)and initiate the apoptotic pathway in cells. Nerve growth factor bindsto two receptors on the cell surface, p75^(NTR) and p140^(trkA). Nervegrowth factor binding to both of these receptors leads to thetransmission of a cell survival signal. If excess amounts of thep75^(NTR) are expressed on cells, more β-amyloid can bind to thep75^(NTR) even in the presence of NGF and initiate the cell deathpathway. If insufficient p140^(trkA) is expressed, which results in anexcess of p75^(NTR) on the cell surface, or if insufficient nerve growthfactor ligand is available to bind to the p75^(NTR), β-amyloid can thenalso bind to the excess p75^(NTR), leading to cell death. All of theabove, or any combination of the above, can lead to apoptosis and AD.

In the proposed diagnostic test, ease of induction of melanocyteapoptosis in vitro following exposure to β-amyloid protein and/or theblocking of this apoptosis by nerve growth factor (NGF) supplementationis correlated with the predisposition of the cell donor to developAlzheimer's disease. Melanocytes obtained from a patient's skin biopsyare compared to standardized control cell lines. β-amyloid protein orpeptide is introduced into the cultures and melanocyte apoptosisresulting from binding of the β-amyloid protein to the p75^(NTR) isdetermined. Activation of the p75^(NTR) by binding of the p75^(NTR) byβ-amyloid protein, or peptide, results in apoptotic cell death of themelanocytes. Thus, activation of the p75^(NTR) by β-amyloid can bemeasured quantitatively as apoptotic cell death of the melanocytes.Apoptotic cell death is readily assessed by any of a number of standardparameters such as propidium iodine incorporation into nuclearfragments, labeling of DNA strand breaks using fluorescein tagged dUTPin the presence of terminal deoxynucleotidyl transferase (TUNELreaction), or by demonstration of fragmented DNA (a DNA ladder).Apoptosis can also be measured by cell counts that reveal progressivecell death. The activation of the p75^(NTR) expressed on the melanocytesobtained from the individual suspected of having AD is compared with theactivation of the p75^(NTR) expressed on control melanocytes. If theactivation of the p75^(NTR) on the individual's melanocytes is greaterthan the activation of the p75^(NTR) on the control melanocytes, it isindicative of the greater risk of the individual to develop Alzheimer'sdisease.

The present invention also relates to methods of treating, or reducingthe risk of developing, Alzheimer's disease, or other neurodegenerativediseases associated with the β-amyloid protein, or by the activation ofthe p75^(NTR) resulting in apoptotic neural cell death. In the proposedtherapy for Alzheimer's disease, a composition containing the tripeptidelysine-glycine, lysine, lysine-glycine-alanine or a similar peptide withestablished affinity for the p75^(NTR) would be delivered by anappropriate route (e.g., intravenously with an agent to reduce theblood-brain barrier or intraventricularly) to central nervous systemneurons at risk of β-amyloid induced apoptotic cell death to blockβ-amyloid binding to p75^(NTR) Nerve growth factor (NGF), biologicallyactive fragments, analogs or derivatives of NGF (wherein biologicalactivity is defined herein as the ability of the fragment, analog orderivative to bind to the p75^(NTR) expressed on neural derived cellssuch as neurons and melanocytes) and/or other neurotropins could also beadministered to further compete against β-amyloid binding to p75^(NTR),as well as to activate cell survival programs within damaged neurons,for example through upregulation of the apoptosis-inhibiting protein,Bcl-2. The therapeutic peptide is designed to have a receptor affinitycomparable to, or greater than, that of β-amyloid but less than that ofNGF and other neurotrophins. Typically, the peptide will be a cyclic, orβ-loop configuration.

Other methods encompassed by the present invention include methods ofdecreasing, or inhibiting the expression of p75^(NTR) by contactingcells expressing the receptor wtih an anti p75^(NTR) anti sense nucleicacid, thereby decreasing or inhibiting the expansion of p75^(NTR) on thecells, thus reducing the risk of β-amyloid mediated apoptosis.Anti-sense p75^(NTR) nucleic acid molecules can be produced by one ofskill in the art using the nucleotide sequence of p75^(NTR) as describedin Johnson, D., et al., Cell,. 47:545-554 (1986).

Alzheimer's disease is a devastating and ultimately fatal disorder.Early detection of Alzheimer's disease would make possible earlyintervention to prevent, or substantially reduce, neuron degenerationand death. Currently, therapies to treat Alzheimer's disease areminimally effective. As effective treatments become available, it wouldpermit rational therapy from an early point in the disease process whensecure diagnosis by conventional clinical criteria is rarely possible.Availability of a therapeutic substance that could slow the progressionof Alzheimer's disease, or other neurodegenerative diseases, in affectedpatients would be a great boon to those individuals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting experimental results showing competition forbinding to p75^(NTR) by peptide versus β-amyloid.

FIG. 2 is a graph depicting experimental results of the effect ofpeptide (SEQ ID NO: 4) on a cell survival in the presence of β-amyloid.

FIG. 3 is a graph showing the results of experiments showing melanocyteapoptosis and survival after treatment with anti p75^(NTR) antisensenucleotide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on Applicants' finding that melanocytesare neural crest-derived cells that share many signal transductionpathways with central nervous system neurons, including the high and lowaffinity receptors for nerve growth factor (Peacocke, M., et al., Proc.Natl. Acad. Sci. USA, 85:5282-5286 (1988); Yaar, M., et al, J. Cell.Biol., 115:821-828 (1991); Yaar, M., et al., J. Clin. Invest.,94:1550-1562 (1994) basic fibroblast growth factor (Halaban, R., et al.,J. Immunother., 12:154-161 (1992), and other molecules. Based on thesesignificant similarities with neurons, it is demonstrated herein thatcultured human melanocytes provide a model system for studies ofAlzheimer's disease.

More specifically, the present invention relates to an in vitro methodof evaluating the risk of an individual to develop Alzheimer's diseasemediated by β-amyloid protein using cultured epidermal melanocytesobtained from the individual. The present invention also relates to theuse of a melanocyte cell culture model or a tissue biopsy sample toevaluate therapeutic compounds effective in the treatment of Alzheimer'sdisease, and other neurodegenerative diseases mediated by β-amyloidprotein. The methods of the present invention are based on Applicants'finding that β-amyloid protein binds to the 75 kilodalton (kD)neurotrophin receptor (p75^(NTR)) (also referred to herein as the lowaffinity nerve growth factor receptor, or p75 nerve growth factorreceptor) which is expressed on the surface of both melanocytes andcentral nervous system neurons, and that when the p75^(NTR) is bound byβ-amyloid, a pathway resulting in cell death (apoptosis) is activated.Applicants further demonstrate herein that this activation can beblocked by substances (e.g., cyclic peptides) that also bind thep75^(NTR), competitively block β-amyloid binding and, hence, preventcell death.

Melanocytes Provide a Model System for Alzheimer's Disease

In vitro studies have demonstrated that β-amyloid protein plays acentral role in Alzheimer's disease. Attempts to understand thepathophysiology of Alzheimer's disease using malignant cell lines(Boland, K., et al, J. Biol. Chem., 270:28022-28028 (1995) or vertebrateneuronal cell cultures (Mark, R. J., et al., J. Neurosci., 15:6239-6249(1995) have revealed that supplementation with β-amyloid peptidescomposed of amino acid residues 1 to 40 or 25 to 35 of the β-amyloidprotein result in neurotoxicity and cell death. In particular, β-amyloidpeptide comprising amino acid residues 1-40 of the β-amyloid protein,amino acid residues 1-42, or, more specifically, amino acid residues25-35 result in neurotoxicity and neuronal cell death. Recent studieshave also shown that β-amyloid-induced neurotoxicity exhibits classicfeatures of apoptosis. The β-amyloid precursor protein (also referred toherein as βAPP) is widely expressed in neural and nonneural mammaliantissues. βAPP is most highly expressed in brain and kidney, and in thebrain, neurons demonstrate particularly high expression of βAPP.

Applicants have demonstrated that human melanocytes and cells ofmelanocytic origin also constitutively synthesize and secrete βAPP.After cellular trauma such as ultraviolet irradiation, there isincreased βAPP secretion, leading to increased βAPP concentration in thevicinity of cultured cells (Andersen, W., et al., J. Invest. Dermatol.,104:585 Abst.182 (April 1995). Such increased βAPP secretion may also bepresumed to occur when neurons are injured.

Applicants demonstrate herein that supplementation of normal humanmelanocytes with β-amyloid 1-40 peptide at low concentrations (≦1 μM)leads to extensive outgrowth of dendrites, the melanocyte cell processesanalogous to neurites of neurons, often a marker of mild injury forwhich the cell can compensate, without decreasing cell yields. At higherconcentrations of β-amyloid peptide, melanocyte cell yields decreaseprogressively and the remaining cells appear unhealthy. Additionally, inthese cultures there is focal development of plaque-like structuresconsisting of aggregated dying melanocytes, similar to the in vivo“senile plaques” observed in patients with AD.

Applicants further demonstrate herein that maintaining melanocytic cellcultures in ≧25 μM β-amyloid 1-40, compared to control cultures,significantly increases the proportion of apoptotic cells andupregulates Bax protein expression approximately three fold. Recent invivo and in vitro data suggest that the β-amyloid induced-neuronal deathexhibits classical characteristics of programmed cell death or apoptosis(Cotman, C. W. and Anderson, A. J., Mol. Neurobiol., 10:19-45 (1995);Su, J. H., et al, Neuroreport, 5:2529-2533 (1994). The molecularpathways that regulate apoptosis in neurons have been identified inpart. Evidence suggests that the product of the proto-oncogene Bcl-2delays the onset of apoptosis in neurons that are dependent for survivalon neurotrophic factors (Allsopp, T. E., et al., Cell, 73:295-307(1993); Garcia, I., et al., Science, 258:302-304 (1992). Conversely,overexpression of a 21 kD Bcl-2-associated protein, Bax, acceleratesapoptotic death of cells (Oltvai, Z. N, et al., Cell, 74:609-619(1993)).

Melanocytes express both the low affinity p75^(NTR) and high affinity140 kD trk A (p140^(trkA)) receptors for NGF. Providing NGF tomelanocytes results in activation of the p140^(trkA) pathway, presumablythrough co-ordinate binding of p140^(trkA) and multiple p75^(NTR)molecules as postulated to occur in neurons exposed to NGF, that in turnactivates an intracellular signal transduction pathway leading toenhanced expression of Bcl-2 and enhanced cell survival. Applicants havealso demonstrated, using melanocytes from newborn, young adult and oldadult donors, that there is a prominent increase in the expression ofthe apoptotic p75^(NTR) with aging.

Applicants demonstrate herein that β-amyloid binds competitively to thep75N. Studies previously published suggest that the specific bindingsite for the p75^(NTR) is amino acids 29-36 of the NGF protein (Ulrich,A., et al., Nature 303:821-825 (1983), and that if the sequencelysine-glycine-lysine (residues 32-34 of NGF) is changed tolysine-glycine-alanine, the peptide has approximately half the affinityfor the receptor as native NGF. Amino acid residues 28-30 of theβ-amyloid protein are lysine-glycine-alanine. Furthermore, computerizedstructure analysis of β-amyloid reveals that these amino acids have ahigh probability of being in a loop turn of the protein, suggesting ahigh probability that this β-amyloid peptide sequence plays a role inreceptor binding.

A cyclic decapeptide was therefore synthesized by attaching two cysteineresidues to the beginning and the end of the β-amyloid fragmentconsisting of amino acids 24-31: VGSNKGAI (SEQ ID NO: 1). Cold peptidecompetitively inhibited ¹²⁵I-β-amyloid binding, with 50% inhibitionoccurring at 25 nM. Furthermore, β-amyloid at a concentration of 200 nMreduced by cell yields by ˜60% (p<0.02), but this cell loss was blockedby the peptide (200 nM). Peptide alone had no effect on cell yield.These findings indicate that apoptosis of neurons in Alzheimer's Diseaseresults from the interaction of β-amyloid with p75^(NTR). The data alsosuggest that β-amyloid-mediated death of neurons may be prevented bydelivery of a synthetic peptide, in the proper conformation, that blocksthe β-amyloid binding sites.

Thus, the above findings of significant similarities of CNS neurons andepidermal melanocytes, permitting their use in diagnostic testing anddrug development studies for Alzheimer's disease.

In Vitro Assay for Alzheimer's Disease

The melanocyte model system described herein can be used for the earlyidentification of individuals who are at risk for developing Alzheimer'sdisease. At present, there are three known or suspected mechanisms forthe development of Alzheimer's disease: 1.) Increased production and/orsecretion of β-amyloid by CNS neurons, a dysfunction known to resultfrom one of the recognized βAPP mutations in familial Alzheimer'sdisease patients; 2.) Increased sensitivity to physiologic β-amyloidconcentration due to excessive local aggregation of the β-amyloidpeptide for as yet unknown reasons, speculated to result from subtlestructural alterations in the secreted β-amyloid or from subtleabnormalities of the extracellular matrix in the CNS; and 3.) Decreasedexpression or function of the low density lipoprotein receptor-relatedprotein on the surface of neurons that is known to bind, internalize,and degrade βAPP and whose functional deficiency would result inincreased amounts of extracellular β-amyloid.

Melanocytes are isolated from skin biopsies obtained from the individualto be tested, i.e., test melanocytes. Skin biopsies are performed usingstandard dermatological techniques. Typically, after application of alocal anesthetic, a 3-4 mm punch biopsy is obtained from the skin of theindividual. Any convenient skin site for biopsy can be selected.Melanocytes isolated from skin are also referred to herein as epidermalmelanocytes.

The melanocytes obtained from the individual are cultured under standardlaboratory conditions as described herein, typically using serum-freemedium. Control cultures of melanocytes are also maintained undersimilar conditions. Control melanocytes can be obtained from anindividual known to be disease-free, from neonatal foreskins, or fromavailable melanocyte cell lines. (See, e.g., Park, H-Y. et al, J. Biol.Chem., 268:11742-11749 (1993)). All melanocytes express the p75^(NTR)plus p140^(trkA) receptor proteins. The cultures are maintained underthese conditions for approximately two days to ensure stable, viablemelanocyte cultures. The cultures can be maintained for longer timeperiods as long as the melanocytes remain viable.

After culture stabilization β-amyloid protein or β-amyloid peptide isintroduced into the cultures. As defined herein, β-amyloid proteinincludes β-amyloid precursor protein as well as β-amyloid protein.β-amyloid peptide includes β-amyloid 1-40 peptide; β-amyloid 1-42peptide; β-amyloid 25-36 peptide and β-amyloid 28-30 peptide. β-amyloidproteins and peptides are commercially available from various sources,e.g, Bachem Calif., Torrance, Calif. β-amyloid protein and peptides canalso be chemically synthesized or recombinantly produced using knownlaboratory techniques. The β-amyloid protein or peptide introduced intothe culture is typically dissolved in a buffer compatible with the cellculture medium. The concentration of β-amyloid protein or peptide addedto the culture can vary from 0 μM to 100 μM, typically from 1 μM to 50μM. A typical single dose of β-amyloid protein or peptide isapproximately 25 μM. The concentration of β-amyloid protein or peptideadded is a concentration sufficient to bind to, and activate thep75^(NTR).

As defined herein, activation of the p75^(NTR) by β-amyloid protein orpeptide means the initiation, or activation of the apoptotic cell deathpathway in the melanocyte. As further defined herein, the activation ofthe p75^(NTR) also means the induction of Bax protein or the initiationof sphingomyelin hydrolysis.

The melanocyte cultures are maintained in the presence of β-amyloidprotein or peptide for a time sufficient for detectable activation ofthe p75^(NTR) to occur, typically about three days. However, the timecan be as short as 1 or as long as 8 days. The activation of thep75^(NTR) of the test melanocytes is compared with the activation of thep75^(NTR) of the control melanocytes.

The activation of the p75^(NTR) by β-amyloid results in apoptotic celldeath of the melanocytes. Apoptosis is determined using well-knownlaboratory techniques. As described herein in Example 1, an assay todetermine melanocyte cell yield was used to determine apoptosis.Apoptosis decreases cell yield. Cell yield is determined by countingviable cells, such as with an electronic cell counting instrument (e.g.,a COULTER™ T cell counter) or by performing a manual cell count using ahemocytometer.

Also as described in Example 1, the cultures can be microscopicallyevaluated for the presence of plaque-like structures containing dying,and/or dead melanocytes. These plaques also contain β-amyloid deposits.

As described in Example 2, activation of the p75^(NTR) can also bedetermined using an assay to measure the inducement of Bax proteinexpression. Bax is the Bcl-2-associated protein implicated in theapoptotic cell death pathway. An increase in the expression of Bax is anindication of apoptosis. Measurement of Bax protein expression can beaccomplished by determining the increase of Bax MRNA expressed in thecells, using standard laboratory techniques to determine RNA in cells.Bax inducement can also be measured using anti-Bax antibody in Westernblot analysis.

Apoptosis can also be determined by the measurement of propidium iodineincorporation into nuclear fragments, the TUNEL reaction, demonstrationof fragmented DNA, or cell count.

If the activation of the p75^(NTR) of the test melanocytes is greaterthan the activation of the p75^(NTR) of the control melanocytes, it isan indication that the individual from whom the test melanocytes wereobtained has neural crest cells that are more sensitive thanstandardized control cell lines to β-amyloid. If the individual's neuralcrest cells are more sensitive to β-amyloid, it is reasonable to predictthat the individual is at risk to develop Alzheimer's disease. Inindividuals whose melanocytes exhibit abnormal sensitivity to β-amyloid,further characterization of the synthesis and deposition ofextracellular matrix molecules can be performed using the melanocyteculture method to determine the interaction of these extracellularmolecules with the β-amyloid peptide.

The cultured melanocyte assay method described herein can also be usefulto evaluate the function of the low density lipoprotein receptor-relatedprotein on the patient's neural crest cells.

Neurons constitutively secrete β-amyloid precursor protein. Becauseβ-amyloid is a major component of the hallmark senile plaques found inAlzheimer's disease patients, the presence of increased levels ofβ-amyloid protein and/or peptide surrounding neurons is reasonablycorrelated with the presence of Alzheimer's disease. Using themelanocyte culture assay method described herein, it can be determinedif the patient's neural crest cells synthesize and/or secrete increasedlevels of β-amyloid constitutively or in response to trauma, in greateramounts as compared to standardized control cell lines. If the secretionof β-amyloid protein or peptide is greater in the test melanocyteculture than in the control melanocyte culture it is indicative that theindividual has an increased risk of developing Alzheimer's disease.

As described above, a number of pathologic mechanisms leading toAlzheimer's disease are known or suspected. As described herein,Applicants demonstrate that the final pathway upon which all the abovemechanisms converge is the activation of the p75^(NTR) alone, leading toapoptosis, instead of the beneficial activation of a receptor complexconsisting of NGF, p75^(NTR) and p140^(trkA). Therefore, thedetermination of the individual's risk developing Alzheimer's diseasecan be accomplished by measuring levels of p75^(NTR) versus p140^(trkA)expressed on the surface of the individual's melanocytes. Individualswith a high ratio of p75^(NTR) to p140^(trkA). A would be those at riskfor developing Alzheimer's disease. Specimens obtained from individualsto be tested can be prepared for immunochemistry, in-situ hybridizationor in-situ PCR, as described herein, to quantitate the expressedreceptors. For example, the method of evaluating the risk of anindividual to develop Alzheimer's disease associated with β-amyloidprotein or β-amyloid peptide activation of the p75 nerve growth factorreceptor comprises the steps of obtaining a skin specimen containingmelanocytes from the individual and processing the specimen to render itsuitable for either immunohistochemistry, in situ hybridization or insitu PCR. For immunohistochemistry, the specimen is contacted with ananti-p75^(NTR) antibody and incubated under conditions suitable for theantibody to bind to the p75^(NTR) present in the melanocytes in thespecimen. The level of bound antibody is measured and therefore thelevel of p75^(NTR) in the specimen is quantified, using methods wellknown in the art. An increase in the level of p75^(NTR) as compared toan age-matched control specimen is an indication that the individual isat risk for Alzheimer's disease.

For in situ hybridization, the specimen is contacted with a labelednucleic acid probe comprising a nucleic acid sequence encoding all or aportion of the p75^(NTR), (Johnson, D., et al., Cell, 47:545-554 (1986))under conditions suitable for hybridization of the probe with nucleicacid encoding p75^(NTR) present in the melanocytes the specimen. Thelevel of hybridized probe, and therefore the level of p75^(NTR) in thespecimen is quantified using methods well known in the art. An increasein the level of p75^(NTR) as compared to an age-matched control specimenis an indication that the individual is at risk for Alzheimer's disease.

For in situ PCR, the specimen is contacted with nucleic acid primerscomprising a nucleic acid sequence encoding the p75^(NTR) underconditions suitable for the amplification nucleic acid encodingp75^(NTR) present in the melanocytes the specimen. The level ofamplified sequence and therefore the level of p75^(NTR) in the specimenis quantified using methods well known in the art. An increase in thelevel of p75^(NTR) as compared to an age-matched control specimen is anindication that the individual is at risk for Alzheimer's disease.

Thus, using the methods described herein, it is reasonable to predictwhether an individual is at risk to develop Alzheimer's disease.Moreover, using the methods of the present invention it is alsoreasonable to predict by what mechanism given individuals are likely todevelop the disease, e.g., abnormal sensitivity β-amyloid, increasedsecretion of β-amyloid protein, or abnormal ratio of expressed p75^(NTR)to p140^(trkA), and thus allow selection of a therapeutic approach thatspecifically targets the abnormality. Furthermore, the method of thepresent invention is useful to screen therapeutic substances, asdescribed below, to determine their effectiveness in an individual.Thus, an individual with either a known predisposition to Alzheimer'sdisease, or a diagnosis of Alzheimer's disease based on conventionalclinical criteria, will be treated with a substance of demonstrated invitro efficacy, and thus, increase chances of alleviating the diseasesymptoms.

Evaluation of Effective Alzheimer's Disease Therapies

NGF binds two receptors: the protein product of the trkA proto-oncogene,p140^(trkA), a transmembrane tyrosine kinase receptor, and a 75 kD lowaffinity transmembrane receptor for several neurotrophins, p75^(NTR).p140^(trkA) has been reported to mediate NGF-induced effects in theabsence of p75^(NTR) (Verdi, J. M., et al., Proc. Natl. Acad. Sci. USA,91:3949-3953 (1994)). It has also been reported that p75^(NTR) mediatesapoptosis of developing neurons in the absence of trkA and thatmodulation of p75^(NGF) receptor can promote neuronal survival(Catharina, F. E., Science, 1725:1729-1732 (Dec. 6, 1996)). However, thefunctional importance of p75^(NTR) in NGF signal transduction is stillcontroversial. It was reported that co-expression of p75^(NTR) andp140^(trkA) produces functional high-affinity binding of NGF (Battleman,D. S., et al., J. Neurosci., 13:941-951 (1993)). It was also reportedthat activation of p75^(NTR) in cells that do not express p140^(trkA)induces their apoptosis by activation of the sphingomyelin signalingpathway. It is therefore possible that p75^(NTR) has a dual role. Incombination with p140^(trkA) it may signal through a tyrosinekinase-dependent pathway (Dobrowsky, T. T., et al., Science, 265:1596(1994) leading to survival, but when activated alone it signals throughthe sphingomyelin pathway leading to apoptosis.

Binding of NGF to p75^(NTR) is mediated through amino acid residues29-36, TDIKGKEV (SEQ ID NO: 2), that are part of the β-hairpin loop ofNGF (Ibánez, C. F., et al., Cell, 69:329-341 (1992)). If lysine (K) atposition 34 is replaced by alanine (A), the resulting mutant NGFmolecule still binds p75^(NTR) but with 50% lower affinity.Interestingly, in β-amyloid the amino acid residues 28-30, which arepresent in both the 1-40 and the 25-35 β-amyloid peptides, are KGA, asequence that appears to permit p75^(NTR) binding by β-amyloid.Computerized structure analysis of β-amyloid suggests that the KGAresidues have a high probability (>60%) of being in a loop turn, thehighest probability of any portion of this 40 amino acid peptide,suggesting that this sequence constitutes a binding site for p75^(NTR).Furthermore, it was reported that expression of p75^(NTR) enhances thetoxic effect of β-amyloid on cells, possibly through binding andactivation of the receptor.

Based on the above data, it is reasonable to believe that the specificthree amino acid sequence lysine-glycine-alanine (KGA) in the β-amyloidprotein binds the 75 kD transmembrane neurotrophin receptor on CNSneurons, activating the programmed cell death pathway, mediated in partby an increase in intracellular Bax levels.

It is also reasonable to believe that competitively inhibiting thebinding of β-amyloid peptide blocks this aberrant receptor activationand the resulting apoptosis. For example, providing full-length NGF, ora biologically active fragment, analog, derivative, variant or mutantthereof results instead in preferential binding of p75^(NTR)coordinately with p140^(trkA) binding, leading to activation of a secondsignal transduction pathway that results in neuronal cell survival. Sucha biologically active NGF fragment would contain the amino acidsequence, lysine-glycine-lysine (KGK).

The term “biological activity” of NGF, or a fragment, derivative,analog, variant or mutant NGF, is defined herein as the activity of theNGF to specifically bind to the p75^(NGF) receptor. Such activity can bemeasured by the methods described herein, or by other methods known tothose skilled in the art. Another biological activity of an NGFfragment, analog, derivative, variant or mutant is the antigenicproperty of inducing a specific immunological response as determinedusing well-known laboratory techniques. For example, a biologicallyactive NGF fragment can induce an immunological response which producesantibodies specific for the NGF (anti-NGF antibodies).

An “analog” is defined herein to mean an amino acid sequence withsufficient identity to amino acid sequence of the endogenous NGF topossess the biological activity of the protein. For example, an analogof a polypeptide can have introduced into the polypeptide amino andsequence “silent” changes wherein one or more amino acid residues differfrom the amino acids residues of the NGF yet still possess p75^(NTR)binding activity. Examples of such differences include additions,deletions, or substitutes of residues. Also encompassed by the presentinvention are proteins that exhibit lesser or greater biologicalactivity of NGF.

The present invention also encompasses the production and use ofbiologically active fragments of the NGF described herein. Suchfragments can include only a part of the full length amino acid sequenceof NGF, yet possess biological activity. As used herein, the term“biologically active fragment” means NGF a fragment that can exert abiological or physiologic effect of the full-length protein, or has abiological characteristic, e.g., antigenicity, of the full-lengthprotein. Such activities and characteristics are described herein. Suchfragments can be produced by amino and carboxyl terminal deletions aswell as internal deletions. Also included are active fragments of theprotein, for example, as obtained by enzymatic digestion. Such peptidefragments can be tested for biological activity.

“Derivatives” and “variants” of NGF is NGF which has been modified. Thisincludes NGF which have been modified by alterations in the amino acidsequence associated with the p57^(NTR). This also includes, but is notlimited to, truncated and hybrid forms of NGF. “Truncated” forms areshorter versions of NGF, for example, modified so as to remove theC-terminal regions. “Hybrid” forms include NGF that is composed ofportions of two or more proteins, i.e., a fusion protein comprising NGFwith another protein.

Variants can be produced using the methods known to those of skill inthe art. The NGF gene can be mutated in vitro or in vivo usingtechniques known in the art, for example, site-specific mutagenesis andoligonucleotide mutagenesis. Manipulations of the NGF sequence can bemade at the protein level as well. Any of numerous chemicalmodifications can be carried out by known techniques including, but notlimited to, specific chemical cleavage by cyanogen bromide, trypsin, andpapain. It can also be structurally modified or denatured, for example,by heat or by being immobilized on a solid surface.

The amino acid sequences of the NGF fragments, analogues, derivatives,variants and mutants of the present invention can be altered to optimizeNGF binding to p75^(NTR), by methods known in the art by introducingappropriate nucleotide changes into native or variant DNA encoding theNGF, or by in vitro synthesis of the desired NGF.

It is reasonable to assume that NGF has a higher affinity for thep75^(NTR) than does β-amyloid, and that when NGF is present in thecellular environment, this neurotrophin preferentially binds thep75^(NTR) coordinately with the p140^(trkA) receptor, activatingp140^(trkA) and leading to cell survival. However, under conditionsleading to increased surface expression of p75^(NTR) or increased levelsof β-amyloid in the extracellular space, in the absence of saturatingamounts of NGF, apoptotic cell death may result from binding ofβ-amyloid to the p75^(NTR) with subsequent activation of thesphingomyelin pathway. In the CNS, such a situation might occur in olderindividuals, particularly those with a genetically-determined excessiveproduction of β-amyloid fragments containing the amino acid sequencerequired for p75^(NTR) binding. Normal human melanocytes appear torespond to environmental signals in the same manner as CNS neurons andreasonably provide a model system for studying potential therapies forAlzheimer's disease.

Thus, the present invention also encompasses methods employingmelanocyte for the development and evaluation of substances to enhancecell survival and to block β-amyloid-induced apoptotic cell death.Specifically, the methods of the present invention can be used toidentify and evaluate substances to treat Alzheimer's disease. Forexample, the melanocyte culture model can be used to identify substancesthat block the p75^(NTR) binding of β-amyloid, and thus blockβ-amyloid-mediated neuronal apoptosis. Several assay methods areavailable in the art to measure p75 nerve growth factor receptoractivation by β-amyloid protein or β-amyloid peptide and include anassay to determine melanocyte cell yield, an assay to determine theinducement of Bax protein expression; an assay to determine the onset ofmelanocyte apoptosis or an assay to determine the presence ofplaque-like structures in the melanocyte culture. Methods to determinethe onset of melanocyte apoptosis are well known in the art and includemeasurement of propidium iodine incorporation into nuclear fragments,the TUNEL reaction or demonstration of fragmented DNA. In addition, inthe method of the present invention, a series of multiple cultures ofthe neural crest-derived cells is maintained; the test substance isadded to each culture such that each culture in the series receives anincreasing concentration of test substance ranging from about 10 μM toabout 100 μM. The activation of the p75 nerve growth factor receptor isdetermined and correlated to the concentration of test substance added.Furthermore, as described in the Examples, inhibition of β-amyloidprotein or β-amyloid peptide binding to the p75 nerve growth factorreceptor can be measured using standard competition assays.

Candidate substances that inhibit the binding of β-amyloidprotein/peptide can also be identified using other cells expressingp75^(NTR). Such cell lines can be engineered to transientially or stablyexpress p75^(NTR) (M. Yaar and B. A. Gilchrest, Arch. Dermatol.,133:1287-1291 (1987).

Furthermore, candidate substances that inhibit the binding of amyloidprotein/peptide can also be identified using in vitro binding assayswell known in the art, such as surface plasmon resonan (SPR). Forexample, peptide libraries and other small organic molecule librariescan be screened using proximity assays or Biospecific InteractionAnalysis (BIA). Biospecific Interaction Analysis (BIA) in real time canbe performed to evaluate candidate molecules for their ability to bindp75^(NTR). Surface plasmon resonance (SPR), which is the basis for BIAmeasurements, is an optical phenomenon arising in metal films underconditions of total internal reflection. The phenomenon produces a sharpdip in the intensity of reflected light at a specific angle. Theposition of this resonance angle depends on several factors, includingthe refractive index of the medium close to the non-illuminated side ofthe metal film. Refractive index is directly related to theconcentration of dissolved material in the medium. By keeping otherfactors constant, SPR is used to measure changes in the concentration ofmacromolecules in a surface layer of solution in contact with adextran-coated gold film. Using the BIACORE™ instrument from PharmaciaBiosensor AB, the association and dissociation rate constants for apeptide or organic molecule binding to p75^(NTR) can be measured.Polypeptides peptides, peptide mimics or small organic moleculesexhibiting higher association constants (K_(a)) have the greatestpotential for ability to interact with p75^(NTR) and inhibit β-amyloidprotein/peptide binding to p75^(NTR).

As described above, the tripeptide lysine-glycine-alanine is a candidatesubstance for use as a therapeutic to alleviate the symptoms ofAlzheimer's disease. The melanocyte culture model system can be used toidentify and evaluate other peptides containing the KGK or KGA sequence,or various analogs of the KGK or KGA tripeptide, to determine an optimumconformation and composition that will bind p75^(NTR) and, thus, blockthe binding of the apoptosis-inducing β-amyloid ligand withoutinterfering with NGF binding that is beneficial. Because evaluation ofcandidate substances is made with melanocytes obtained specifically fromthe individual at risk, or with the diagnosis of AD, the probability ofidentifying an effective candidate substance is very high.

Once candidate substances are identified, the therapeutic level ofsubstance that must be delivered to the CNS can be accurately determinedusing in vitro melanocyte cultures from each individual. Titration ofconcentration of the candidate substance can be performed using themelanocyte culture model system described herein.

Also encompassed by the present invention are methods of therapy for thetreatment of neurodegenerative diseases resulting from apoptosis due tothe aberrant activation of the p75^(NTR) receptor of neuralcrest-derived cells, or increased expansion of the p75^(NTR).Specifically encompassed are methods of therapy for individualsexperiencing symptoms of dementia resulting from degeneration, anddeath, of neurons due to β-amyloid-mediated apoptosis.β-amyloid-mediated apoptosis is a hallmark of Alzheimer's disease,therefore, also specifically encompassed by the present invention aremethods of treating individuals with Alzheimer's disease.

Methods of therapy include administering to the individual a substance,e.g., a cyclic peptide comprising KGK or KGA, or an analog thereof, in amanner which permits contact of the substance with neurons of the CNS.For example, the pentapeptide CKGAC (SEQ ID NO: 3), or an analogthereof, can be chemically synthesized by methods well-known to one ofskill in the art. The cysteine residues flanking the ends of thepentapeptide can be linked, e.g., by a disulfide bond, to maintain theconformation required for binding of the peptide to the p75^(NTR), thusinhibiting, or preventing apoptosis. The length of the peptide can belonger than a pentapeptide, as long as the KGA, or analog peptide ismaintained in a configuration suitable for binding activity. Forexample, as described herein, cyclic peptides have been made with theamino acid sequences and CVGSNKGAIC (SEQ ID NO: 4) these peptidescompete for p75^(NTR) binding with β-amyloid peptide. It is reasonableto believe that other cyclic peptides would also bind p75^(NTR) andinhibit apoptosis. Administration, or delivery of the peptide, or othersubstance, can be accomplished in a manner similar to methods used ingene transfer and therapy. For example, an effective amount of the DNAencoding the peptide can be inserted into a virus vector construct whichtargets central nervous system neurons. The DNA insert also includessequences necessary for the expression of the DNA in the target cells.Particularly useful is the Herpes Simplex

Virus-1 (HSV-1) vector described in Geller, A. I., and Breakefield, X.O., Science 241:1667-1669 (1988) and U.S. Pat. No. 5,288,641 (Roizman1994), the teachings of these references are herein incorporated byreference. Gene guns can also be used. Additionally, intracranialadministration can also be employed.

Further in vivo testing cam be performed using an art-recognized mousemodel such as the transgenic mouse described in WO 96/40895, theteachings of which are incorporated herein by reference. Such testing iswell-known to those of skill in the art.

The following examples more specifically illustrate the invention andare not intended to be limiting in any way.

EXAMPLE 1 The Effect of β-amyloid on Normal Human Melanocytes

To determine the effect of β-amyloid on normal human melanocytes,cultures were supplemented with increasing concentrations (0.025-50 uM)of HPLC-purified β-amyloid fragment corresponding to amino acids 1-40. Apeptide containing the first 40 amino acids synthesized in reverse order(40-1) was used as a negative control.

Melanocytes were maintained in serum free Medium 199 (Gibco BRLGaithersburg, Md.) supplemented with epidermal growth factor (10 ng/ml)(Collaborative Research), insulin (10 μg/ml) (Sigma), triiodothyronine(10⁻⁹ M) (Collaborative Research), transferrin (10 μg/ml) (Sigma),hydrocortisone (1.4×10⁻⁶ M) (Calbiochem), cholera toxin (10⁻⁹ M)(Calbiochem) and basic fibroblast growth factor (basic FGF) (10 ng/ml)(Collaborative Research). Two days after plating cells were supplementedwith increasing concentrations of β-amyloid 1-40 or the control peptide40-1 (0-50 μM) (Bachem Calif., Torrance, Calif.). Cell yields determinedthree days after addition of β-amyloid show a dose dependent decrease ofcell yield in cultures maintained with the 1-40 peptide. No effect oncell yield was observed in cultures maintained with the control 40-1peptide.

Melanocytes maintained in the presence of 25-30 uM β-amyloid 1-40 showeda 59%±17% decrease in cell yield as compared to cell yield beforeβ-amyloid addition that is considered to be 100%. Cell yield ofduplicate cultures maintained in the presence of the control 40-1peptide showed 8%±32% increase in cell yield (p<0.02, paired t test).

Melanocytes maintained in the presence of the 40-1 control peptide havea typical bipolar to polygonal morphology. The majority of themelanocytes maintained in the presence of the 1-40 peptide are roundedand detaching from the dish surface.

Regression analysis showed significant decrease in cell yield withincreasing concentrations of β-amyloid 1-40 (R²=0.8475, p<0.00001) butno significant effect on cell yield of β-amyloid 40-1 (R²=0.06, p=0.44).In a total of four experiments, within 3-5 days β-amyloid 1-40 decreasedmelanocyte yield by >50% (p<0.02; paired t test) while the control 40-1β-amyloid peptide at the same concentrations had no effect on cellyield.

EXAMPLE 2 Effect of β-amyloid 1-40 on Melanocyte Plaque Formation

Melanocyte cultures, cultured as described above in Example 1, were alsoevaluated for plaque formation. In some cultures the development ofplaque-like structures consisting of increasingly large congregations ofdying melanocytes was noted, reminiscent of the senile plaques describedin the brains of patients with Alzheimer's disease.

EXAMPLE 3 Effect of β-amyloid and NGF on Melanocytes

In neurons, the protein product of the proto-oncogene Bcl-2 delays theonset of apoptosis triggered by a variety of stimuli, whileoverexpression of a Bcl-2 associated protein (Bax) accelerates this celldeath.

To investigate the mechanism of β-amyloid-mediated melanocyte death, Baxlevels in melanocytes treated with 25 uM of 1-40 or 25-35 β-amyloidpeptides was examined. Within 4 days of treatment, Bax was induced 3fold in melanocytes stimulated either with the β-amyloid 1-40 or 25-35fragments as compared to melanocytes treated with the 40-1 controlfragment or an irrelevant HPLC purified protein of similar size.

Melanocytes were maintained as above. Four days after addition of 25 μMof β-amyloid fragments 1-40, 40-1 or 25-35; or 25 μM of HPLC-purifiedbovine corticotropin releasing factor (CRF) (Bachem Calif.) (MW 4.7 kD)as an additional negative control, cells were extracted in RIPA buffer(50 mM Tris-HCl, 0.15 M NaCl, 0.5% sodium deoxycholate, 1% TRITON®X-100)in the presence of 1 μ/ml aprotinin and 75 μg/ml phenylmethylsulfonylfluoride (PMSF), sonicated for 1-3 seconds and centrifuged. 40 μg ofprotein per lane were separated on 12% SDS/PAGE and blotted ontonitrocellulose paper (overnight, 25V). To verify equal loading aduplicate 13% SDS/PAGE was performed and stained with Coomasie Blue R250stain. Blots were incubated with anti-Bax antibody (1:1000 dilution)(primary antibody), followed by horseradish peroxidase-conjugated goatanti-rabbit IgG (secondary antibody) (1:500 dilution) (Bio-RadLaboratories, Hercules, Calif.). Bound antibodies were detected usingenhanced chemiluminescence kit (Amersham Corp.). Autoradiograms werescanned into a Macintosh II is computer using the Of to TM program(Light source Computer Images, Inc.). Scan analysis was performed withmanual definition of bands using Scan Analysis TM 68000 program(Biosoft, Cambridge, UK).

β-amyloid peptides 1-40 and 25-35 increased Bax level as compared to the40-1 peptide (270% and 160%, respectively).

EXAMPLE 4 Effect of NGF on β-amyloid Induced Cell Death

Nerve growth factor (NGF) has been reported to improve cognitivefunction and attenuate loss of cholinergic neurons in animal models andin clinical treatment trials for Alzheimer's disease (Seiger, A., etal., Behave. Brain. Res., 57-255-261 (1993); Lapchak, P. A., Exp.Neurol., 124:16-20 (1993); Olson, L., et al., J. Neural. Transm. Park.Dis. Dement. Sect., 4:79-95 (1992)). Furthermore, it has been recentlyreported that NGF delays melanocyte apoptosis by upregulating the levelsof Bcl-2 (Zhai, S., et al. Exp. Cell. Res. It was investigated whetherNGF supplementation protects melanocytes from p-amyloid-induced celldeath. Addition of NGF to β-amyloid-supplemented melanocytes enhancedcell yields within 3-5 days and greatly improved the morphology ofsurviving cells in most donors, although degree of protection wasvariable among donors. Preliminary data suggest that NGF supplementationdecreases the β-amyloid-induced Bax upregulation and increases Bcl-2levels in the cells suggesting that NGF interferes withβ-amyloid-mediated signal transduction.

Melanocytes were maintained as above in hormone supplemented mediumlacking hydrocortisone. Cells were supplemented with 25 μM of β-amyloid1-40 in the presence of 50 ng/ml NGS or diluent.

Photographs of representative fields were obtained and the percent ofthe live cells (spread) was determined 48 hours after addition ofβ-amyloid and NFG or β-amyloid and diluent. In the presence of β-amyloidand diluent 77±8.5% appeared spread as compared to 96±1.4% in culturessupplemented in β-amyloid and NGF. In cultures not provided β-amyloidand diluent even the cells that were still spread on the dish surfacewere vacuolated and generally did not appear as healthy as cells in NGFsupplemented cultures.

β-amyloid exposed melanocytes in the absence of NGF are dying, while inthe presence of NGF the cells appear healthy and spread on the dishsurface. At least 400 cells were counted in each condition.

EXAMPLE 5 β-amyloid Binds the p75 Nerve Growth Factor Receptor

To determine if β-amyloid binds p75^(NTR), ¹²⁵I labeled 1-40 β-amyloidpeptide was added to permanently transfected cultured fibroblasts thatoverexpress p75^(NTR) (p75^(NTR)-NIH 3T3 cells) (Dobrowsky, T. T., etal., Science, 265:1596 (1994) in the presence of disuccinimidyl suberateto cause cross linking of closely associated proteins. Cells were thenimmunoprecipitated with anti p75^(NTR) antibodies or an irrelevant mouseIgG. Autoradiograms revealed a protein band of 75-80 kD in size only inlysates immunoprecipitated with anti p75^(NTR) antibodies. Competitionanalysis of ¹²⁵I 1-40 β-amyloid in the presence of increasingconcentrations of unlabeled NGF showed that 1-40 β-amyloid could becompeted off by NGF. However, residual ¹²⁵I labeled 1-40 bindingsuggests that 1-40 β-amyloid may have an additional cell surfacereceptor, perhaps the recently identified serpin-enzyme complexreceptor.

Results indicate that p75^(NTR) is a receptor for β-amyloid, a peptidethat is reported to be secreted into the medium of normal cells in highpicomolar to low nanomolar concentrations.

p75^(NTR)-NIH 3T3 cells were maintain in DMEM supplemented with 10% FBSin the presence of penicillin (45 ng/ml), streptomycin (68 ng/ml), andhygromycin B (17.5 ng/ml). At 80% confluence cells were lifted from thedish with EDTA and incubated in suspension with 5 uCi ¹²⁵I labeledβ-amyloid 1-40 at 4° C. for 1 hour in DMEM. After incubation, 1 mM ofdisuccinimidyl suberate was added for 30 minutes. Followingcentrifugation cells were lysed with RIPA buffer (50 mM Tris HCl, pH8.0, 0.15 M NaCl 0.5% sodium deoxycholate 4, 5 mM MgC_(2,) 1%TRITON®X-100, 1 mM phenylmethylsulfonylfluoride, and 1 μg/ml aprotinin),sonicated for 1-3 seconds and immunoprecipitated with anti p75^(NTR)antibodies (mouse monoclonal IgGl, Cedarlane Laboratories Ltd., Ontario,Canada) or mouse IgG as control for 16 hours at 4° C. in the presence of15 ul of protein G plus protein A agarose and 1M NaCl adjusted to pH8.0. After several washes with 20 mM Tris HCl, pH 8.0, 1M NaCl, 5mMMgCl₂, 0.2% TRITON®X-100 and 1 mM PMSF, immunoprecipitates wereseparated over 8% PAGE and subjected to autoradiography. A band ofappropriate molecular weight was present only in lysatesimmunoprecipitated with anti p75^(NTR) antibodies and not lysatesimmunoprecipitated with irrelevant mouse IgG.

p75^(NTR)-NIH 3T3 cells were incubated for 2 hours at 4° C. in bindingmedium (DMEM, 10 mM hepes, 0.1 mg/ml cytochrome C, 0.01% TWEEN®80, 1mg/ml BSA) with ¹²⁵I labeled β-amyloid 1-40 and increasingconcentrations of NGF (0-100 ng/ml). After rinsing in PBS, cells werelysed in 1N NaOH and equal amounts of protein from cell lysates weresubject to counting. A concentration dependent inhibition of ¹²⁵Ilabeled β-amyloid binding by NGF was observed with a maximum of 38%inhibition at NGF concentrations of 100 ng/ml and statisticallycomparable binding at 25 ng/ml.

EXAMPLE 6 Cyclic Peptide Competitively Inhibits β-amyloid Binding top75^(NTR)

p75^(NTR) 3T3 cells were incubated in suspension at 4° C. for 4 hourswith 0.5 uCi ¹²⁵I labeled β-amyloid 1-40 and increasing concentrations(0-400 nM) of the cyclic peptide CVGSNKGAIC (SEQ ID NO: 4). Lysates from1.5×10⁵ cells were subjected to counting. As shown in FIG. 1,concentration dependent inhibition of ¹²⁵I labeled β-amyloid 1-40binding by the cyclic peptide with 50% inhibition was observed at theexpected 25 nM cyclic peptide concentration. This experimentdemonstrates the cyclic peptide can compete with β-amyloid 1-40 forbinding to the p75^(NTR) receptor.

EXAMPLE 7 Effect of Peptide on Cell Survival

p75^(NTR) 3T3 cells were maintained in serum free medium supplementedwith 200 nM β-amyloid 1-40, 200 nM cyclic peptide CVGSNKGAIC, (SEQ IDNO: 4) 20 nM β-amyloid 1-40 and 200 nM cyclic peptide or diluent alone.Cell yields determined up to 120 hours after addition of β-amyloid showprominent decrease of cell yield in cultures maintained with 200 nMβ-amyloid 1-40 as compared to all other cultures (see FIG. 2).

EXAMPLE 8 Methods for Testing the Level pf p75^(NTR) mRNA and Protein inSkin Specimens

Skin specimens will be obtained from early postmortem Alzheimer'sdisease patients and age matched controls. Specimens will be embeddedand frozen in Tissue Tek II O.C.T. compound and processed forimmunohistochemistry, in-situ Hybridization, and in-situ PCR.

Immunohistochemistry

4-6 um thick vertical sections of skin will be incubated 1st with FITCconjugated mouse anti p75^(NTR) monoclonal antibody as described (Yaar,M., et al., Lab. Invest., 58:175-162 (1988)). Then skin specimens willbe incubated with Rhodamine conjugated anti TRP-1 antibody, a marker forcutaneous melanocytes. The intensity of anti-p75^(NTR) antibody bindingin melanocytes will reflect the level of p75^(NTR). Quantification willbe performed using a computer image analyzer (Allan, A. E., et al., J.Invest. Dermatol., 105:678-692 (1995)).

In-situ Hybridization

Riboprobe Preparation

The p75^(NTR) cDNA will be cloned into a transcription vector pGEM-3Zcontaining both the T7 and PS6 polymerase promoter sites. Transcriptswill be tested and the transcript that has been correct orientation thatis complementary to p75^(NTR) mRNA will be labeled with 35D-UTP (Allan,A. E., et al., J. Invest. Dermatol., 105:678-692 (1995)). 3 um sectionswill be cut, applied to glass slides and hybridized with the labeledprobe. After extensive washes the slides will be dipped in liquidemulsion and placed at 4° C. for 10 days (Yaar, M., et al., Exp. CellRes., 17:217-226 (1995)). At the end of the exposure period the slideswill be developed and stained with hematoxylin and eosin. Slides will beviewed under light- and dark-field illumination and intensity of thelabeling will be quantified using a computer image analyzier (Allan, A.E., et al., J. Invest. Dermatol., 105:678-692 (1995)).

In-situ PCR

In-situ PCR will be performed as described in Nuovo et al. (Nuovo, G.J., et al., Am. J. Pathol. 139:1239-1244 (1991)). Backward and forwardprimers spanning a 115 bp sequence of the coding region of p75^(NTR)mRNA will be generated. Primers that span an intron will be selected toeliminate the possibility of amplifying genomic DNA. We will usereagents provided by the Gene Amp core kit from Perkin-Elmer and followthe instructions of the manufacturer Nuovo et al. (Nuovo, G. J., et al.,Am. J. Pathol. 139:1239-1244 (1991), Khan, Z. M., et al., J. InvestDermatol., 106:667-672 (1996). 45 cycles of PCT amplification will beperformed. After hybridization, slides will be incubated with anti TRP-1antibodies and a secondary goat anti mouse IgG. Color detection will becarried out using the substrates 4-nitroblue tetrazolium and5-bromo-4-chloro-3-indolyl phosphate. Samples will be counterstainedwith hematoxylin and eosin and viewed under light field illumination(Khan, Z. M., et al., J. Invest Dermatol., 106:667-672 (1996)). Theintensity of p75^(NTR) mRNA labeling will be quantified using a computerimage analyzer.

EXAMPLE 9 The Dual Function of the p75^(NTR) in Melanocyte Apoptosis andSurvival

Melanocytes were UV irradiated with 25 mJ/cm² and then supplementedeither with 10 μM anti p75^(NTR) anti sense phosphothioateoligonucleotides (CTCCCACTCGTCATTCGAC SEQ ID NO.: 5) directed againstthe 3′ end of the p75^(NTR) coding region, or the same concentration ofsense oligonucleotides as a negative control. Half the cultures wereprovided with 50 ng/ml NGF and; half the cultures were provided diluentalone.

Immunohistochemical analysis confirmed that p75^(NTR) was not expressedin cells treated with anti-p75^(NTR) anti-sense oligonucleotides, butwas expressed in the sense treated cells. Within 24 hours afterirradiation; the following was observed the following were observed:

1. Cells expressing p75^(NTR) (supplemented with sense oligonucleotides)and trk A and supplemented with NGF appear healthy (A), establishing theprotective effect of NGF on melanocyte survival in the presence of bothreceptors.

2. The majority of the cells expressing p75^(NTR) (supplemented withsense oligonucleotides) and trk A and supplemented with diluent insteadof NGF are dying as apparent by their rounded morphology (B).

3. Cells not expressing p75^(NTR) (supplemented with anti-senseoligonucleotides) but expressing trk A and provided with 50 ηg/ml NGF(C) do not appear as healthy or numerous as cells expressing bothreceptors in the presence of NGF (A), establishing that the presence ofboth NGF receptors is required for optimal NGF effect.

4. Cells not expressing p75^(NTR) (supplemented with anti-senseoligonucleotides) although they were not provided NGF (D) appear ashealthy as cells provided NGF lacking p75^(NTR) (C) establishing thatp75^(NTR) is required to induce melanocyte apoptosis.

Equivalents

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that the various changes in form and detailsmay be made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211> LENGTH: 8<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1Val Gly Ser Asn Lys Gly Ala Ile  1               5 <210> SEQ ID NO 2<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 2 Thr Asp Ile Lys Gly Lys Glu Val  1               5<210> SEQ ID NO 3 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Cyclic peptide <400> SEQUENCE: 3Cys Lys Gly Ala Cys  1               5 <210> SEQ ID NO 4<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Cyclic peptide <400> SEQUENCE: 4Cys Val Gly Ser Asn Lys Gly Ala Ile Cys  1               5  #                10 <210> SEQ ID NO 5 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide <400> SEQUENCE: 5ctcccactcg tcattcgac              #                  #                   # 19

What is claimed is:
 1. A method of identifying a substance that inhibitsβ-amyloid-mediated apoptosis of neural crest-derived cells, comprisingthe steps of: a) culturing neural crest-derived cells that express p75nerve growth factor receptor; b) introducing into the cell culture ofstep a) a polypeptide selected from the group consisting of: β-amyloidprecursor protein, β-amyloid protein, and β-amyloid peptide, in aconcentration sufficient to bind to and activate the p75 nerve growthfactor receptor, and the substance to be tested, thereby producing atest-culture; c) maintaining the test-culture of step b) underconditions sufficient for the polypeptide or test-substance to bind tothe p75 nerve growth factor receptor expressed on the neuralcrest-derived cells; d) determining activation of the p75 nerve growthfactor receptor; and e) comparing activation of the p75 nerve growthfactor receptor of the neural crest-derived cells cultured in step b)with activation of the p75 nerve growth factor receptor of neuralcrest-derived cells in a control culture comprising cells cultured inthe absence of the substance, wherein a decrease of activation of thep75 nerve growth factor receptor of neural crest-derived cells in thetest culture compared to activation of the p75 nerve growth factorreceptor of neural crest-derived cells in the control culture isindicative of a substance that inhibits β-amyloid-mediated apoptosis inneural crest-derived cells.
 2. The method of claim 1, whereindetermining p75 nerve growth factor receptor activation furthercomprises an assay method selected from the group consisting of: anassay to determine cell yield, an assay to determine the inducement ofBax protein or mRNA expression; an assay to determine the onset ofapoptosis or an assay to determine the presence of plaque-likestructures in the culture.
 3. The method of claim 2, wherein the assayto determine the onset of apoptosis is an assay selected from the groupconsisting of: measurement of propidium iodide incorporation intonuclear fragments, the TUNEL reaction or demonstration of fragmentedDNA.
 4. The method of claim 1, wherein the β-amyloid peptide is selectedfrom the group consisting of: β-amyloid 1-40 peptide; β-amyloid 1-42peptide; β-amyloid 25-35 peptide or β-amyloid 28-30 peptide.
 5. Themethod of claim 1, wherein the concentration of the polypeptide in stepb) is from about 0.025 μM to about 100 μM.
 6. The method of claim 1,wherein in step a) a series of cultures of the neural crest-derivedcells is maintained; in step b) the test substance is added to eachculture such that each culture in the series receives an increasingconcentration of test substance ranging from about 25 nM to about 100 μMand in step d) the activation of the p75 nerve growth factor receptor isdetermined and correlated to the concentration of test substance added.7. The method of claim 1 wherein the neural crest-derived cells aremelanocytes.
 8. A method of identifying a substance that inhibitsbinding of a polypeptide selected from the group consisting of:β-amyloid precursor protein, β-amyloid protein, and β-amyloid peptide top75 nerve growth factor receptor expressed by melanocytes, comprisingthe steps of: a) culturing melanocytes that express the p75 nerve growthfactor receptor; b) introducing into the cell culture of step a) apolypeptide selected from the group consisting of: β-amyloid precursorprotein, β-amyloid protein, and β-amyloid peptide, in a concentrationsufficient to bind to and activate the p75 nerve growth factor receptor,and the substance to be tested, thereby producing a test-culture; c)maintaining the test-culture of step b) under conditions sufficient forthe polypeptide or test-substance to bind to the p75 nerve growth factorreceptor expressed on the melanocytes; d) determining activation of thep75 nerve growth factor receptor; and e) comparing activation of the p75nerve growth factor receptor of melanocytes cultured in step b) withactivation of the p75 nerve growth factor receptor of melanocytes in acontrol culture comprising cells cultured in the absence oftest-substance, wherein a decrease of activation of the p75 nerve growthfactor receptor of melanocytes in the test culture compared toactivation of the p75 nerve growth factor receptor of melanocytes in thecontrol culture is indicative of a substance that inhibits β-amyloidbinding to the p75 nerve growth factor receptor of melanocytes.
 9. Themethod of claim 8, wherein in step a) a series of cultures ofmelanocytes is maintained; in step b) the test substance is added toeach culture such that each culture in the series receives an increasingconcentration of test substance ranging from about 25 nM to about 100 μMand in step d) the activation of the p75 nerve growth factor receptor isdetermined and correlated to the concentration of test substance added.10. A method for identifying a substance that inhibitsβ-amyloid-mediated apoptosis of cells, wherein the cells are fibroblastcells transfected to express p75 nerve growth factor receptor,comprising the steps of: a) measuring p75 nerve growth factor receptoractivation in a sample of the cells exposed to polypeptide selected fromthe group consisting of: β-amyloid precursor protein, β-amyloid protein,and β-amyloid peptide, wherein the polypeptide is present at aconcentration sufficient to bind and activate the p75 nerve growthfactor receptor, and wherein the sample of cells is also exposed to thesubstance to be tested, thereby producing a test culture; b) measuringp75 nerve growth factor receptor activation in a sample of the cellsexposed to polypeptide selected from the group consisting of: β-amyloidprecursor protein, β-amyloid protein, and β-amyloid peptide in theabsence of the substance to be tested, wherein the polypeptide is at aconcentration sufficient to bind and activate the p75 nerve growthfactor receptor, thereby producing a control culture; and c) comparingthe activation of the p75 nerve growth factor receptor of the test andthe control cultures, wherein a decrease of activation of the p75 nervegrowth factor receptor in the test culture compared to the controlculture is indicative of a substance that inhibits β-amyloid-mediatedapoptosis in the cells, thereby identifying a substance that inhibitsβ-amyloid-medicated apoptosis of cells.
 11. The method of claim 10,wherein activation of p75 nerve growth factor receptor is determinedusing a method selected from the group consisting of: an assay todetermine cell yield, an assay to determine induction of Bax protein ormRNA expression; an assay to measure apoptosis and an assay to determinethe presence of plaque-like structures in the sample.
 12. The method ofclaim 11, wherein the assay to measure apoptosis is selected from thegroup consisting of: measurement of propidium iodide incorporation intonuclear fragments, TUNEL reaction, and measurement of fragmented DNA.13. The method of claim 10, wherein the concentration of polypeptide isfrom about 0.025 μM to about 100 μM.
 14. The method of claim 10, whereina series of test cultures is maintained and wherein the test substanceis added to each culture such that each culture in the series receivesan increasing concentration of test substance ranging from about 25 nMto about 100 μM and wherein the activation of the p75 nerve growthfactor receptor is determined and correlated to the concentration oftest substance added.
 15. The method of claim 10, wherein the cells areNIH 3T3 cells.
 16. A method for determining whether a substance inhibitsβ-amyloid-mediated apoptosis of cells, wherein the cells are transfectedwith a p75 nerve growth factor receptor gene such that the cells expressp75 nerve growth factor receptor, comprising the steps of: a) measuringp75 nerve growth factor receptor activation in a sample of the cellsexposed to a polypeptide selected from the group consisting of:β-amyloid precursor protein, β-amyloid proteins, and β-amyloid peptide,wherein the polypeptide is present in a concentration sufficient to bindto and activate the p75 nerve growth factor receptor, and wherein thesample of cells is also exposed to the substance to be tested, therebyproducing a test culture; b) measuring p75 nerve growth factor receptoractivation in a sample of the cells exposed to the polypeptide in theabsence of the substance to be tested, wherein the polypeptide is in aconcentration sufficient to bind and activate the p75 nerve growthfactor receptor, thereby producing a control culture; and c) comparingthe activation of the p75 nerve growth factor receptor of the test andthe control cultures, wherein a decrease of activation of the p75 nervegrowth factor receptor in the test culture compared to the controlculture is indicative of a substance that inhibits β-amyloid-mediatedapoptosis in cells.
 17. A method for determining whether a substanceinhibits β-amyloid-mediated apoptosis of cells, wherein the cells areneural crest-derived cells that express p75 nerve growth factorreceptor, comprising the steps of: a) measuring p75 nerve growth factorreceptor activation in a sample of the cells exposed to a polypeptideselected from the group consisting of: β-amyloid precursor protein,β-amyloid protein and β-amyloid peptide, wherein the polypeptide ispresent in a concentration sufficient to bind to and activate the p75nerve growth factor receptor, and wherein the sample of cells is alsoexposed to the substance to be tested, thereby producing a test culture;b) measuring p75 nerve growth factor receptor activation in a sample ofthe cells exposed to the polypeptide in the absence of the substance tobe tested, wherein the polypeptide or peptide is in a concentrationsufficient to bind and activate the p75 nerve growth factor receptor,thereby producing a control culture; and c) comparing the activation ofthe p75 nerve growth factor receptor of the test and the controlcultures, wherein a decrease of activation of the p75 nerve growthfactor receptor in the test culture compared to the control culture isindicative of a substance that inhibits β-amyloid-mediated apoptosis inneural crest-derived cells.
 18. The method of claim 17, wherein theneural crest-derived cells are melanocytes.